Here we show that Bmp signaling is necessary and sufficient for the specification of ventral endoderm in Xenopus embryos. Overexpression of Bmp4 in ectoderm induces markers of endoderm, including Sox17β, Mixer and VegT, but cannot induce the expression of the dorsoanterior markers, Xhex and Cerberus. Furthermore, knockdown approaches using overexpression of Bmp antagonists and morpholinos designed against Bmp4, Bmp2 and Bmp7 demonstrate that Bmp signaling is critical for ventral, but not dorsoanterior endoderm formation. This activity is not simply a result of embryonic dorsalization as markers for dorsal endoderm are not expanded. We further show that no endodermal cells of either ventral or dorsal character form when both Wnt and Bmp signals are abolished. Overall, this report strongly suggests that Bmp plays an essential role in ventral endoderm specification.

The transcription factors Mixer and Sox17␤ have well-characterized roles in endoderm specification during Xenopus embryogenesis. In order to more thoroughly understand the mechanisms by which these endodermal regulators act, we expressed Mixer and Sox17␤ in naïve ectodermal tissue and, using oligonucleotide-based microarrays, compared their genomic transcriptional profile to that of unaffected tissue. Using this approach, we identified 71 transcripts that are upregulated by Mixer or Sox17␤, 63 of which have previously uncharacterized roles in endoderm development. Furthermore, an in situ hybridization screen using antisense probes for several of these clones identified six targets of Mixer and/or Sox17␤ that are expressed in the endoderm during gastrula stages, providing new and regional markers of the endoderm. Our results contribute further insight into the functions of Mixer and Sox17␤ and bring us closer to understanding at the molecular level the pathways that regulate endoderm development.

This study describes a cross-species functional screen of mouse gastrula cDNA libraries for components of endoderm and mesoderm specification. Pools of 96 cDNAs from arrayed mouse gastrula cDNA libraries were transcribed into mRNA and injected into either the presumptive mesoderm or the ectoderm of one-cell Xenopus laevis embryos. Injected embryos were examined at gastrula stage by in situ hybridization with endoderm or mesoderm markers. Using this approach, we screened over 700 pools or ∼60,000 cDNAs. We identified 17 unique cDNAs that function during mesoderm and/or endoderm specification and 16 that cause general morphology changes. Identified molecules fall into eight general functional groups as follows: cell cycle components (seven), transcription factors (four), extracellular secreted molecules (seven), transmembrane receptors (one), intracellular signaling components (five), microtubule components (two), metabolism molecules (three), and unknown (four). Several of the genes we identified would not have been predicted to be involved in endoderm or mesoderm specification, highlighting the usefulness of nonbiased screening approaches. This includes Otx2, which we show is a downstream target of Xsox17␤. The speed, low cost, and high efficiency of this cross-species screen makes it an ideal method for examining cDNAs from difficult-to-obtain sources. Therefore, this approach complements the current mouse molecular genetics systems and provides a powerful means for the genome-wide examination of mammalian gene function.[The sequence data from this study have been submitted to dbEST/GenBank under accession nos.

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